growthdifferentiationfactor(gdf)-15blocksnorepinephrine ...trophic effects have been characterized...

Growth Differentiation Factor (GDF)-15 Blocks Norepinephrine-induced Myocardial Hypertrophy via a Novel Pathway InvolvingInhibition of Epidermal Growth Factor Receptor Transactivation*

Received for publication, September 6, 2013, and in revised form, February 11, 2014 Published, JBC Papers in Press, February 19, 2014, DOI 10.1074/jbc.M113.516278

Xin-ye Xu, Ying Nie, Fang-fang Wang, Yan Bai, Zhi-zhen Lv, You-yi Zhang, Zi-jian Li1, and Wei Gao2

From the Department of Cardiology, Peking University Third Hospital and Key Laboratory of Cardiovascular Molecular Biology andRegulatory Peptides, Ministry of Health, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education and BeijingKey Laboratory of Cardiovasicular Receptors Research, Beijing 100191, China

Background: The effect of GDF-15 on the regulation of cardiac remodeling is poorly understood.Results: GDF-15 inhibits norepinephrine induced EGF receptor transactivation and associated hypertrophy and positivelyassociates with myocardial hypertrophy of hypertension patients.Conclusion: GDF-15 blocks norepinephrine-induced myocardial hypertrophy via a novel pathway involving inhibition of EGFreceptor transactivation.Significance: We indicate a negative feedback mechanism of GDF-15 in regulating catecholamine-induced cardiac remodeling.

Accumulating evidence suggests that growth differentiationfactor 15 (GDF-15) is associated with the severity and prognosisof various cardiovascular diseases. However, the effect ofGDF-15 on the regulation of cardiac remodeling is still poorlyunderstood. In this present study, we demonstrate that GDF-15blocks norepinephrine (NE)-induced myocardial hypertrophythrough a novel pathway involving inhibition of EGFR transac-tivation. Both in vivo and in vitro assay indicate that NE was ableto stimulate the synthesis of GDF-15. The up-regulation ofGDF-15 feedback inhibits NE-induced myocardial hypertrophy,including quantitation of [3H]leucine incorporation, protein/DNA ratio, cell surface area, and ANP mRNA level. Furtherresearch shows that GDF-15 could inhibit the phosphorylationof EGF receptor and downstream kinases (AKT and ERK1/2)induced by NE. Clinical research also shows that serum GDF-15levels in hypertensive patients were significant higher than inhealthy volunteers and were positively correlated with the thick-ness of the posterior wall of the left ventricle, interventricular sep-tum, and left ventricular mass, as well as the serum level of norepi-nephrine. In conclusion, NE induces myocardial hypertrophy andup-regulates GDF-15, and this up-regulation of GDF-15 negativelyregulates NE-induced myocardial hypertrophy by inhibiting EGFreceptor transactivation following NE stimulation.

Growth differentiation factors (GDFs)3 are a subfamily ofproteins belonging to the transforming growth factor � (TGF�)

superfamily that have functions predominantly in development(1). Several members of this subfamily have been described andnamed GDF-1 through GDF-15. GDF-15, also known asMIC-1, NAG-1, and PLAB, has been identified as a biphasicregulator of cell survival and inflammation in several patholog-ical processes including injury and tumorigenesis (2, 3).

In recent years, an increasing number of investigations havefocused on the association between GDF-15 and cardiovasculardiseases. Normally, GDF-15 is expressed at very low levels inhealthy heart, but its expression can be significantly up-regu-lated in myocardial injury, ischemia, and cardiac remodeling.GDF-15 has proved valuable in the prognosis of coronary heartdisease and heart failure (4 –13). Recent studies showed thatGDF-15 was involved in the process of cardiac remodeling,including hypertrophic cardiomyopathy, primary hyperten-sion, and ischemic heart diseases (13–17). Our previous dataalso indicated that GDF-15 may be involved in the compensa-tory stage of the pathological process of heart failure (12). Theseresults suggested that GDF-15 could play a role in the process ofmyocardial hypertrophy. So far, both pro- and anti-hyper-trophic effects have been characterized (18, 19); however,whether GDF-15 acts as an injury factor or a protector, aswell as its precise function and mechanism of action,remains unclear.

Sustained activation of the sympathetic nervous system leadsto alterations in normal myocardial structure (20). Previousresults indicated that the endogenous adrenergic agonist nor-epinephrine (NE) was responsible for the hypertrophic processin vivo and in vitro (21). The regulatory features of NE-inducedhypertrophy were highly complex, however, and numeroussubstances have been implicated (22–24). Whether GDF-15might be a novel regulatory factor in NE-induced myocardialhypertrophy is still unknown.

In this research, we analyzed the relationships betweenGDF-15 and clinical features of myocardial hypertrophy, as well

* This work was supported by Grant PUCRP-004 from the Key ConstructionProgram of the National 985 Project, National Natural Science Foundationof China Grants 30910103902, 81270157, 81070078, and 81121061, Grantfrom the 973 Program of the Major State Basic Research DevelopmentProgram of China 2011CB503903, and Beijing Municipal Natural ScienceFoundation Grant 7102158.

1 To whom correspondence may be addressed. E-mail: [email protected] To whom correspondence may be addressed. E-mail: [email protected] The abbreviations used are: GDF, growth differentiation factor; NE, norepi-

nephrine; EGFR, EGF receptor; LVPW, left ventricle posterior wall; IVS, inter-ventricular septum; LVMI, left ventricular mass index; NRCM, neonatal rat

cardiomyocyte; ANP, atrial natriuretic peptide; BNP, type B natriuretic pep-tide; �-MHC, � myosin heavy chain; AR, adrenergic receptor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 14, pp. 10084 –10094, April 4, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

10084 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 14 • APRIL 4, 2014

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as between GDF-15 and NE. To explain the clinical relation-ships observed, the study focused on elucidating (i) whether theGDF-15 level could be affected by NE stimulation and (ii) thepotential role of GDF-15 in NE-induced myocardial hypertro-phy and its molecular mechanisms.

EXPERIMENTAL PROCEDURES

Clinical Analysis—A cohort study was conducted betweenJuly 2009 and August 2011 in the Department of Cardiology ofPeking University Third Hospital (Beijing, China). A total of 63consecutive inpatients, who were diagnosed with primaryhypertension based on the European Society of Cardiologyguidelines for the management of arterial hypertension (32),and 30 healthy volunteers, were enrolled. The cohort was testedfor a correlation of serum GDF-15 level with echocardiographitems and the serum level of NE, together with other clinicalparameters, to investigate the relationship between GDF-15and myocardial hypertrophy. Two-dimensional echocardiog-raphy was performed with an Vivid 7 cardiac ultrasound unit(GE, VINGMED). Left ventricular hypertrophy was assessed bymeasurement of the left ventricle mass and the thickness of theposterior wall of left ventricle (LVPW) and interventricularseptal (IVS). Mitral inflow was assessed in the apical four-chamber view, using pulsed-wave Doppler echocardiography,with the Doppler beam aligned parallel to the direction of flowand the sample volume at the leaflet tips. From the mitral inflowprofile, the E wave was measured. Doppler tissue imaging of themitral annulus was obtained from the apical four-chamber view,using a 1–2-mm sample volume placed in the septal mitral valveannulus. Left ventricular mass index (LVMI) and body surface area(BSA) were calculated based on the following formulas,

LVmass � 0.8[1.04(IVSTd � LVIDd � LVPWTd)]3 � 0.6

(Eq. 1)

BSA(male) � 0.0057 � Height � 0.0121 � Weight � 0.0882

(Eq. 2)

BSA(female) � 0.0073 � Height � 0.0127 � Weight � 0.2106

(Eq. 3)

LVMI � LVmass/BSA (Eq. 4)

where LVmass is left ventricular mass. All the blood sampleswere obtained at 8:00 on an empty stomach. Serum GDF-15and NE levels were measured by ELISA (GDF-15: ELH-GDF15– 001 RayBiotech, Inc.; NE: H21662; BioASSAY Co.,Ltd. Hong Kong, China). Both GDF-15 and NE were measuredaccording to the manufacturer’s instructions.

Patients who developed cardiac shock or severe congestiveheart failure (NYHA III-IV) were excluded from the study.Other exclusion criteria were acute myocardial infarctionwithin 12 weeks, impairment of renal function, systemic in-flammatory disease, infectious diseases, cancer, acute cerebralinfarction, prostate diseases, pregnancy, or use of a steroid thatmight affect the serum GDF-15 levels.

This study was approved by the Medical Ethics Committee ofPeking University, and all subjects provided written informed

consent. The study was conducted in compliance with the Dec-laration of Helsinki.

Animals and Drug Treatment—The investigation was ap-proved by the Biomedical Research Ethics Committee of PekingUniversity (LA 2010-048) and adhered to the American Physi-ological Society’s Guiding Principles in the Care and Use ofVertebrate Animals in Research and Training. One-day-oldneonatal and 10-week-old adult Sprague-Dawley rats were pro-vided by the Animal Department of Peking University HealthScience Center (Beijing, China). All experimental protocolswere approved by the Peking University Institutional Commit-tee for Animal Care and Use. The rats were hypodermicallyinjected with norepinephrine twice per day with a total dose of2.4 mg/kg/day or ascorbic acid (control) for 7 days. The ratswere weighed, and hearts were excised and then rinsed in PBSbefore use.

Preparation of Cardiomyocytes—Cardiomyocytes were iso-lated and cultured from 1-day-old neonatal Sprague-Dawleyrats as described previously (28). In brief, neonatal rat car-diomyocytes (NRCMs) were grown in DMEM (Invitrogen)with 10% fetal bovine serum (Hyclone) and antibiotics (50mg/ml streptomycin, 50 units/ml penicillin) at 37 °C in a 5%CO2 atmosphere. 5-BrdU (100 �mol/l) was added to the cul-ture medium to prevent proliferation of non-myocardio-cytes. NRCMs were starved overnight in DMEM mediumwithout serum before use in experiments.

ELISA—GDF-15 protein levels in the serum and supernatantwere measured using a commercially available ELISA kitaccording to the manufacturer’s instructions. All samples wereassayed in triplicate.

Western Blot—Cell lysates were subjected to SDS-PAGE andblotted on nitrocellulose membranes. Membranes were thenincubated with antibodies directed against GDF-15, phospho-Akt (Ser-473), phospho-ERK1/2 (Thr-202/Tyr-204), and phos-pho-EGFR (Tyr-1173) and then against the total level of eachprotein, respectively (all the above antibodies were from CellSignaling Technology Inc., Danvers, MA). Protein bands werevisualized with the Gel Logic 212 imaging system (Kodak,Rochester, NY).

Immunohistochemistry—Hearts were excised and fixed with4% paraformaldehyde overnight and embedded in paraffin. Tis-sue morphometry was evaluated in a blinded fashion using theLeica Q550 IW imaging work station (Leica MicrosystemsImaging Solutions Ltd., Cambridge, UK). GDF-15 expressionwas revealed with diaminobenzidine staining (primary anti-body: GDF-15 (E-19) antibody, sc-10607; Santa Cruz Biotech-nology, Inc.). The number of positively stained (brown) cellswas expressed as a percentage of total area.

Real Time PCR—Total RNA was extracted by using TRIzol(Invitrogen). One microgram of total RNA was then reversetranscribed into cDNA by use of the RT-PCR access kit (Invit-rogen). One microliter of cDNA was used for PCR. Rat atrialnatriuretic peptide (ANP), B-type natriuretic peptide (BNP), �myosin heavy chain (�-MHC), and GAPDH mRNA wereamplified by PCR. The PCR mixture was incubated in a DNAThermal Cycler (Stratagene, La Jolla, CA). The reactions wereprepared in triplicate and heated to 95 °C for 5 min, followed by40 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. The

GDF-15 in Transactivation-dependent Myocardial Hypertrophy

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resulting relative increase in reporter fluorescent dye (SYBRGreen) emission was monitored with the use of the ABI PRISM7700 sequence detector (Applied Biosystems).

Adenovirus Transfection—Recombinant adenovirus strainsexpressing EGFR (pAV-EGFR-eGFP), GDF-15 (pAV-GDF15-eGFP), �-arrestin1 (pAV-�-arrestin1-eGFP), or EGFP shRNAwere generated by subcloning the cDNA encoding EGFR, GDF-15, or �-arrestin1 into the vector pAd/CMV/V5-DESTTM

Gateway� and EGFR shRNA, GDF-15 shRNA, or �-arrestin1shRNA into the pAV5� U6-shRNA adenoviral vector. NRCMswere incubated with recombinant virus strains for 24 h beforeexperimentation.

Immunofluorescence—NRCMs were treated with NE (10 �M)for 0 , 5, or 15 min; then fixed with 4% paraformaldehyde; per-meabilized with 0.2% Triton X-100 in PBS; and incubated withanti-EGFR (Tyr-1173) antibody (diluted 1:100) at 4 °C over-night and FITC-conjugated anti-rabbit IgG antibody (diluted1:400, Santa Cruz Biotechnology) at 37 °C for 1 h. For actinstaining, cardiomyocytes were also stained with phalloidin (5�g/ml) at 37 °C for 30 min after permeabilization for cell sizeanalysis. After washing with PBS, NRCMs were examinedunder an inverted fluorescence microscope (DM IRE2; Leica,Wetzlar, Germany). Cell size was measured by Image-Pro Plus6.0 (Media Cybernetics Inc.).

[3H]Leucine Incorporation—After preincubation with orwithout GDF-15, cardiomyocytes were treated with NE (10 �M)

for 48 h in serum-free medium and further incubated for 6 hwith 1 �Ci/ml [3H]leucine before harvesting. Protein was col-lected by 10% trichloroacetic acid precipitation, resuspended in1 M sodium hydroxide, and analyzed using a liquid scintillationcounter (LS 1801; Beckman Coulter Inc.). The radioactivity ineach sample was normalized against protein concentration.

Statistics—All values are expressed as means � S.D. Thesample size in each group is denoted by n. An unpaired t testwas used to assess the differences in GDF-15 levels betweenpatients and healthy volunteers. Pearson’s correlations and lin-ear regressions were used to evaluate the relationship ofGDF-15 to different clinical parameters and echocardiographymeasurements. One-way analysis of variance followed by theNewman-Keuls test was used for multiple comparisons.GraphPad Prism (GraphPad Software, San Diego, CA) softwarewas used for statistical analysis.

RESULTS

NE Stimulates GDF-15 Secretion—To investigate the rela-tionship between GDF-15 and NE in the induction of myocar-dial hypertrophy, NRCMs were stimulated with NE in theconcentration range 1–100 �M. GDF-15 levels increased signif-icantly in both NRCM supernatant (Fig. 1A) and cell lysate (Fig.1B) in a dose-dependent manner after incubation with NE.GDF-15 concentration in the supernatant peaked at 24 h afterNE (10 �M) stimulus and remained at high level over the next

FIGURE 1. NE induced GDF-15 expression in NRCMs. A, ELISA showing the GDF-15 levels in supernatant induced by NE in different concentrations for 24 h,the peak level of GDF-15 appeared when treated with NE (100 �M). B, Western blot showing the GDF-15 expression in cell lysate induced by NE in differentconcentrations for 24 h; the peak level of GDF-15 appeared when treated with NE (100 �M). C, ELISA showing the GDF-15 levels in supernatant induced atdifferent time points after NE (10 �M) stimulation; the peak level of GDF-15 appeared 24 h after NE treatment. D, Western blot showing the GDF-15 level in celllysate at different time points after NE (10 �M) stimulation; the level of GDF-15 increased significantly 24 h after NE treatment. Con, control; Vitc, vitamin C. *, p �0.05; n � 5.




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24 h (Fig. 1C). GDF-15 concentration in cell lysate also peaked24 h after NE(10 �M) stimulus and showed a slightly downwardtendency in the next 24 h (Fig. 1D).

Similar results were observed in experiments with Sprague-Dawley rats. An ELISA revealed that serum GDF-15 levelsincreased significantly when rats were injected with NE at aconcentration of either 1.2 or 2.4 mg/kg/day. The serum level ofGDF-15 reached a maximum 24 h after NE injection and thendeclined to baseline after 3 days (Fig. 2, A and B). Western blotalso revealed increased GDF-15 level in cardiac tissue after NEinjection (Fig. 2C). Further, immunohistochemistry assay illus-trated the maximum expression of GDF-15 in rat cardiac tissueat the time point of 24 h after the NE injection (Fig. 2D). Theseresults suggested that the synthesis and secretion of GDF-15could be stimulated by NE in either cardiomyocytes or at thewhole animal level.

GDF-15 Negatively Regulates NE-induced MyocardialHypertrophy—To investigate the functional effects of in-creased GDF-15 in NE induced myocardial hypertrophy,NRCMs were stimulated with NE (10 �M) in the presence orabsence of GDF-15 for 48 h. ANP, BNP, and �-MHC are knownto be up-regulated during the process of myocardial hypertro-phy (35). Our results revealed that GDF-15 (20 ng/ml) signifi-

cantly reduced the effect of NE on ANP, BNP, and �-MHCmRNA levels (Fig. 3, A–C). Further, because it is considered thegolden standard of myocardial hypertrophy, the level of cardiacmyocyte protein synthesis was also analyzed by measurementsof [3H]leucine incorporation and total protein/DNA ratio.Treatment in isolated cardiomyocytes with NE resulted in anincrease in protein/DNA ratio (Fig. 3D) and [3H]leucine incor-poration (Fig. 3E), both of which were inhibited by preincuba-tion with GDF-15. NE stimulation resulted in more [3H]leucineincorporation in NRCMs with GDF-15 knockdown (Fig. 3F). Inaddition, the increased cell surface area of cardiomyocytesinduced by NE was also inhibited by GDF-15 (20 ng/ml) (Fig.3G). Taken together, these results indicate that GDF-15negatively regulates NE-induced myocardial hypertrophy andtherefore likely functions as a sympathetic regulator.

GDF-15 Negatively Regulates NE-induced Transactivation ofEGFR and Its Signaling Pathway—The kinases Akt and ERKhave previously been shown to play an essential role in myocar-dial hypertrophy (33, 34). Western blot revealed the inhibitoryeffect of GDF-15 preincubation upon the phosphorylation ofAkt and ERK induced by NE (Fig. 4, D–F). Data from our pre-vious study and other recent studies indicated that adrenergicreceptor (AR) agonists were capable of inducing transactivation

FIGURE 2. NE induced GDF-15 expression in Sprague-Dawley rats. A, ELISA showing the GDF-15 levels in rat serum induced by NE in different doses. Thelevel of serum GDF-15 elevated 3 h after NE injection, maintained a high level in 24 h, and declined to baseline in 72 h. B, ELISA revealed increased serum GDF-15levels when treated with NE (both 1.2 and 2.4 mg/kg/day) for 12 h. C, Western blot revealed increased tissue GDF-15 levels when treated with NE (both 1.2 and2.4 mg/kg/day) for 12 h. D, immunohistochemistry tissue section illustrating more GDF-15 expression in rat cardiomyocytes 24 h after NE (2.4 mg/kg/day)stimulation. Con or con, control, *, p � 0.05; n � 3.




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of EGFR (28, 29). Considering that Akt and ERK both act down-stream of EGFR, we hypothesized that NE transactivates EGFRand that GDF-15 probably inhibits the activation of EGFR. Ourresults revealed that NE significantly induced EGFR transacti-vation with immunofluorescence assay (Fig. 4A) and Westernblot (Fig. 4, D–F) in cardiomyocytes, which could be inhibitedby preincubation with GDF-15 (Fig. 4, D–F). Similar resultswere also observed in experiments with Sprague-Dawley rats.Immunochemistry assay and Western blot revealed thatGDF15 (1 �g/kg) mediated inhibition of EGFR transactivationinduced by NE treatment (Fig. 4, B and C). Furthermore, ade-novirus-mediated knockdown of GDF-15 or EGFR wereemployed to confirm the inhibitory effect of GDF-15 upon NE-induced transactivation of EGFR. The phosphorylation ofEGFR and its downstream signaling (Akt and ERK) inducedby NE were significantly enhanced in NRCMs with GDF-15knockdown alone (Fig. 4, G–I), whereas these enhancement

could be abolished in NRCMs with co-knockdown of GDF-15and EGFR (Fig. 4, J–L). Taken together, these results showedthat GDF-15 inhibits the NE-induced phosphorylation of EGFRand its downstream kinases, Akt and ERK, thus implicatingGDF-15 in a novel NE-induced EGFR transactivation pathway.

Adenovirus strains for EGFR overexpression was also con-structed and introduced into NRCMs, to further elucidate theantagonistic effect of GDF-15 on NE-induced transactivation ofEGFR. The phosphorylation of EGFR (Fig. 5A), ERK (Fig. 5B),and Akt (Fig. 5C) induced by NE in EGFR-overexpressing car-diomyocytes was significantly augmented compared with con-trols without EGFR overexpression (p � 0.05). In addition,[3H]leucine incorporation experiments showed that NE-in-duced protein incorporation was enhanced in EGFR-overex-pressing cardiomyocytes (Fig. 5G) and was attenuated in EGFRknockdown cardiomyocytes (Fig. 5H). These results suggestthat transactivation of EGFR is responsible for NE-induced

FIGURE 3. GDF-15 inhibited NE-induced myocardial hypertrophy. A–C, RT-PCR showed that GDF-15 inhibited the increase of relative ratios of ANP (A), BNP(B), and �-MHC (C) mRNA levels induced by NE (10 �M). D, GDF-15 inhibited the increase of protein-DNA ratio induced by NE (10 �M). E, less [3H]leucineincorporation induced by NE was observed in GDF-15 preincubated NRCMs than in NRCMs without GDF-15 incubation. F, more [3H]leucine incorporationinduced by NE was observed in GDF-15 knockdown NRCMs than in NRCMs without the knockdown of GDF-15. G, F-actin staining showed that GDF-15 inhibitedthe enlargement of cell surface area induced by NE (10 �M) with contributing analysis. Con, control; CPM, counts per minute. *, p � 0.05 n � 4.




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FIGURE 4. GDF-15 inhibited the phosphorylation of EGFR induced by NE in NRCMs. A, immunofluorescence illustrating that NE (10 �M) was able toinduced EGFR phosphorylation. B, immunochemistry assay revealed that intravenous injection of GDF-15 (1 �g/kg) significantly attenuated the EGFRphosphorylation induced by NE stimulation. C, Western blot revealed that GDF-15 inhibited the phosphorylation of EGFR induced by NE in tissue. D–F,less phosphorylation of Akt (D), ERK1/2 (E), and EGFR (F) induced by NE was observed in GDF-15 preincubated NRCMs than in NRCMs without GDF-15incubation. G–I, more phosphorylation of Akt (G), ERK1/2 (H), and EGFR (I) induced by NE was observed in GDF-15 knockdown NRCMs than in NRCMswithout the knockdown of GDF-15. J–L, less phosphorylation of Akt (J), ERK1/2 (K), and EGFR (L) induced by NE was observed in NRCMs with theknockdown of GDF-15 alone than in GDF-15/EGFR co-knockdown NRCMs. Con, control; Scr, scrambled; Vitc, vitamin C. *, p � 0.05 n � 3.




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FIGURE 5. GDF-15 inhibited NE induced transactivation of the EGFR signaling pathway and t myocardial hypertrophy. A–C, Western blot showedthat the phosphorylation of EGFR (A), ERK1/2 (B), and Akt (C) induced by NE and the extent of inhibition by GDF-15 were more significant in NRCMs withEGFR overexpression than in NRCMs without overexpression. D–F, Western blots showing that the phosphorylation of EGFR (D), ERK1/2 (E), and Akt (F)induced by NE were reduced in EGFR knockdown NRCMs than in Scr, and no inhibition of phosphorylation by GDF-15 was observed. G, more [3H]leucineincorporation induced by NE was observed in EGFR-overexpressed NRCMs than in NRCMs without EGFR overexpression. The [3H]leucine incorporationof NRCMs with or without EGFR overexpression was inhibited by GDF-15 to a similar level. H, less [3H]leucine incorporation induced by NE was observedin EGFR knockdown NRCMs than in NRCMs without EGFR knockdown. GDF-15 could not inhibit NE-induced [3H]leucine incorporation in NRCMs withEGFR knockdown. Scr, scrambled; CPM, counts per minute. *, p � 0.05; n � 3.




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myocardial hypertrophy. Moreover, GDF-15 blocked NE-stim-ulated EGFR transactivation (Fig. 5A) and further phosphor-ylation of the downstream kinases ERK (Fig. 5B) and Akt (Fig.5C). GDF-15 also blocked EGFR transactivation-mediatedmyocardial hypertrophy ([3H]leucine incorporation) (Fig. 5G),and this effect was abolished in EGFR knockdown cardiomyo-cytes (Fig. 5H). In contrast, NE failed to induce phosphor-ylation of EGFR (Fig. 5D), ERK (Fig. 5E), and Akt (Fig. 5F) inEGFR knockdown cardiomyocytes. The blockage of EGFRtransactivation by GDF-15 was also abolished in EGFRknockdown cardiomyocytes (Fig. 5, D–F). Our findings dem-onstrate that GDF-15 is a novel negative regulator of NE-in-duced myocardial hypertrophy through inhibition of NE-in-duced EGFR transactivation.

Because �-arrestin1 was involved in NE induced EGFR trans-activation and myocardial hypertrophy (28, 29), we assessedwhether �-arrestin1 mediated the negative regulation of NE-induced EGFR transactivation by GDF-15. The results revealedthat the knockdown of GDF-15 enhanced phosphorylation ofEGFR, ERK, and Akt, which were abolished in �-arrestin1-de-ficient cardiomyocyte (Fig. 6, A–C). These data demonstratedthat the inhibitory effect of GDF-15 upon the EGFR transacti-vation induced by NE was �-arrestin1-dependent.

GDF-15 Levels Positively Correlate with EchocardiographItems and Norepinephrine Concentrations in Serum—To fur-ther analyze the clinical significance of GDF-15 in cardiachypertrophy in hypertension, a total of 63 hypertensive patientsand 30 volunteers joined our study. There was no age differencebetween patients and volunteers (62.79 � 14.43 versus 58.26 �5.99, t � 1.649, p � 0.103). The serum GDF-15 levels in hyper-tensive patients ranged from 894.85 to 1505.20 pg/ml, with amean value of 1193 � 163.4 pg/ml, which was significantlyhigher than the GDF-15 levels of healthy volunteers (308.58 �239.371 pg/ml, p � 0.01) (detailed clinical and echocardiographdata not shown here). Patient GDF-15 levels significantly andpositively correlated with parameters contributing to myocar-dial remodeling including BNP (r � 0.2786, p � 0.05) and echo-cardiograph parameters such as thickness of IVS (r � 0.4503,p � 0.01), LVPW (r � 0.7103, p � 0.01), left atrial pressure (r �0.3906, p � 0.01), and mitral early diastolic flow versus earlydiastolic peak velocity (E/Em) (r � 0.3731, p � 0.01). Bothserum levels of NE and GDF-15 were positively correlated withLVMI (Table 1 and Fig. 7). These results demonstrated a tightrelation between GDF-15 and myocardial hypertrophy and the

relation between myocardial hypertrophy and activation of thesympathetic nervous system. Further, a significant positive cor-relation between GDF-15 and serum levels of NE, an inducer ofmyocardial hypertrophy, was also demonstrated (r � 0.7334,p � 0.01), which indicated that GDF-15 could be involved inactivation of the sympathetic nervous system.

DISCUSSION

Our previous studies indicated that serum GDF-15 concen-tration was elevated with increasing stage of heart failure andmight have potential value in distinguishing stage B heart fail-ure (12). The clinical value of GDF-15 as a biomarker in cardio-vascular diseases was also demonstrated in other studies.GDF-15 was not only identified as a prognostic marker of cor-onary heart disease and heart failure, where higher serumGDF-15 levels are associated with worse outcomes (4 –9), butwas also valuable in guiding the selection of treatment (4). Fur-ther, GDF-15 was implicated in pathological processes such asthe development of atherosclerosis and angiogenesis of lateralarteries (11). However, the relationship between GDF-15 andmyocardial hypertrophy has been examined only infrequently.A recent study demonstrated that the serum level of GDF-15elevated significantly in hypertension patients with left ventric-ular hypertrophy (36). Our current study revealed that GDF-15positively correlated with IVS and LVPW at the prehypertro-phy stage of hypertension, as well as left atrial pressure andexcitation/emission, which indicated that GDF-15 was proba-bly involved in the compensatory stage of myocardial hypertro-phy. Further laboratory data indicated that GDF-15 negatively

FIGURE 6. �-Arrestin1 mediated the negative regulation of NE-induced EGFR transactivation by GDF-15. Less phosphorylation of EGFR (A), Akt (B), andERK1/2 (C) induced by NE was observed in NRCMs with GDF-15/�-arrestin1 co-knockdown than in NRCMs with GDF-15 knockdown alone. *, p � 0.05; n � 3.

TABLE 1GDF-15 levels of hypertension patients in relation to biochemical andechocardiography measuresThe p value was calculated from the regression analysis with GDF-15 as the depen-dent variable. US-CRP, ultrasensitive-C reactive protein; E/Em, excitation/emis-sion; LAP, left atrial pressure; Rho, Pearson’s correlation coefficient.

Value (n � 63) Rho p

Biochemical parametersBNP (pg/ml) 541.0 � 79.0 0.2786 p � 0.05Triclycerides (mmol/liter) 1.716 � 0.8647 �0.2730 p � 0.05US-CRP (mmol/liter) 4.106 � 1.92 0.3045 p � 0.05NE (pg/ml) 135.9 � 17.72 0.7334 p � 0.01

Echocardiography measuresIVS (mm) 9.3 � 2.1 0.4503 p � 0.01LVPW (mm) 8.5 � 1.4 0.7103 p � 0.01E/Em 9.5 � 4.9 0.3731 p � 0.01LAP (mm Hg) 13.0 � 6.1 0.3906 p � 0.01LVMI (g/m2) 82.6 � 25.3 0.5572 p � 0.01




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regulated NE-induced myocardial hypertrophy, which wasconsistent with the observation in a transverse abdominal aor-tic constriction model that GDF15 antagonized pressure over-load-induced hypertrophy (18). These results are consistentwith a role for GDF-15 as a protective factor in myocardialhypertrophy.

Chronic sympathetic activation is known to be a key factor inthe progression of myocardial hypertrophy, with NE as animportant endogenous agonist of the sympathetic system (20).Clinical data from our research demonstrated a positive corre-lation between serum GDF-15 and NE levels. Furthermore, ourlaboratory data indicated that this relationship was not just aconcomitant phenomenon, but a cause and effect chain ofevents. Continuous treatment with NE was found to induce thesynthesis and secretion of GDF-15 in myocardium, both at thecellular and whole animal levels. Previously, various exogenousagents such as cyclooxygenase inhibitors, dietary agents, PPARagonists, and some anti-cancer drugs were shown to induceGDF-15 synthesis (3). Our work is the first to identify an endog-enous stimulator of GDF-15 and to suggest a potential molec-ular network involving GDF-15 and catecholamines.

The data we present suggest a model whereby up-regulationof GDF-15 inhibits NE-induced myocardial hypertrophy byacting as a protective agent in a negative feedback loop with NE(Fig. 8). A similar protective effect has been reported in otherconditions including ischemia/reperfusion injury and infarc-tion, as well as pressure-induced hypertrophy (18, 25, 26). Inprevious studies, several endogenous agents have been identi-fied as sympathetic regulators, including catestatin, spinophi-lin, syntrophins, and angiotensin II: syntrophin regulates�1-AR through a PDZ domain-mediated interaction (22), spi-

nophilin stabilizes cell surface expression of �2-AR and blocksarrestin1 action on GPCR (23), and catestatin interferes withthe level of catecholamine and the activation of phospholipaseC (24). Here, we identified GDF-15 as a novel sympathetic reg-ulator that protects against myocardial hypertrophy throughrepression of NE-induced transactivation of EGFR.

Multiple pathways are involved in AR-related myocardialhypertrophy, with the transactivation of EGFR representing anovel pathway demonstrated in recent years (27). We showed

FIGURE 7. A, positive correlation between GDF-15 and NE (r � 0. 7334, p � 0.01). B, positive correlation between GDF-15 and LVPW (r � 0.7103, p � 0.01). C,positive correlation between GDF-15 and IVS (r � 0.4503, p � 0.01). D, positive correlation between GDF-15 and LVMI (r � 0.5572, p � 0.01). E, positivecorrelation between NE and LVMI (r � 0.7264, p � 0.01).

FIGURE 8. Working model of critical role for GDF-15 in NE-induced myo-cardial hypertrophy. GDF-15 synthesis and secretion could be stimulated byNE both in vivo and in vitro. Increased GDF-15 in turn inhibited NE-inducedmyocardial hypertrophy by inhibiting NE-induced transactivation of EGFRand the phosphorylation of downstream kinases Akt and ERK, which con-structed a feedback manner.




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previously that �-AR-induced transactivation of EGFR maylead to myocardial hypertrophy (28); �-AR-related EGFR trans-activation was also demonstrated (29). As a major endogenousagonist of AR, NE simultaneously activates both the �- and�-AR and induces myocardial hypertrophy. The demonstra-tion that NE can induce myocardial hypertrophy throughtransactivation of EGFR and its downstream kinases, Akt andERK, and that GDF-15 can inhibit this process is the first reportto our knowledge of a mechanism for the inhibition of thetransactivation pathway of EGFR.

It has been demonstrated that �-arrestin1 was involved as amediator in the process of transactivation. The �-arrestin1recruitment was essential in adrenergic receptor-mediatedEGFR transactivation (28, 29). Our results revealed that theknockdown of GDF-15 enhanced the phosphorylation of EGFRinduced by NE, whereas co-knockdown of GDF-15 and �-ar-restin1 abolished this enhancement, indicating that the inhibi-tory effect of GDF-15 upon the transactivation induced by NEwas �-arrestin1-dependent.

Interestingly, the true effect of GDF-15 on myocardial hyper-trophy remains controversial. GDF-15 was found to inhibitmyocardial hypertrophy through a Smad2/3 pathway in a pres-sure-induced hypertrophy model (18), whereas directly treat-ing cultured cardiomyocytes with GDF-15 results in pro-hyper-trophic effect through a Smad1 pathway (19). It seems that theexact effect differs in different conditions. Previous studiesmainly focused on the Smad-dependent mechanism, which wasconsidered the canonical signaling pathway of GDF-15. How-ever, the Smad pathway has not been proved to be so importantin the hypertrophic model induced by sympathetic activation.Here, our data suggested that GDF-15 may protect the heartfrom NE-induced hypertrophy through a Smad-independentpathway.

In the recent guidelines for heart failure, several new phar-macotherapies including BNP, simendan, and ivabradine weresuggested in clinical use (30). Although new pharmacothera-pies have developed rapidly in recent years, �-blockers,together with angiotensin-converting enzyme inhibitors, werestill considered the cornerstones for heart failure treatment andreversing cardiac remodeling. Our study demonstrated anotherpotential role of �-blockers, and potentially also angiotensin-converting enzyme inhibitors, in the reversal of remodeling andhypertrophy by inhibition of the EGFR transactivation path-way. Finally, it seems clear that GDF-15, as a new modulator ofsympathetic system, should not be considered purely as a bio-marker, but also as a potential new pharmacotherapy for heartfailure and cardiac remodeling.

CONCLUSION

Beyond its prognostic value of numerous cardiovascular dis-eases, GDF-15 secretion is also tightly correlated with the pro-cess of myocardial hypertrophy and chronic activation of thesympathetic nervous system in hypertensive patients. Investi-gations of function and molecular mechanisms demonstrated afeedback loop in which the synthesis of GDF-15 could be stim-ulated by NE in cardiomyocytes, and in turn, elevated GDF-15could negatively regulate NE-induced hypertrophy. GDF-15was identified as a new endogenous sympathetic regulator that

restricts the transactivation of EGFR NE (Fig. 8). Our resultssuggested that GDF-15 could be a new therapeutic target forthe treatment of myocardial hypertrophy.

Acknowledgment—We thank Alan Tunnacliffe for critical evaluationof the manuscript.

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and Wei GaoXin-ye Xu, Ying Nie, Fang-fang Wang, Yan Bai, Zhi-zhen Lv, You-yi Zhang, Zi-jian Li

Growth Factor Receptor TransactivationMyocardial Hypertrophy via a Novel Pathway Involving Inhibition of Epidermal

Growth Differentiation Factor (GDF)-15 Blocks Norepinephrine-induced

doi: 10.1074/jbc.M113.516278 originally published online February 19, 20142014, 289:10084-10094.J. Biol. Chem.

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growthdifferentiationfactor(gdf)-15blocksnorepinephrine ...trophic effects have been characterized...

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